Angiogenesis is a fundamental process by means of which new blood vessels are formed. This process is essential in many normal physiological phenomena such as reproduction, development and cicatrization. In these normal biological phenomena, angiogenesis is under strict control, i.e., it is triggered during a short period of several days then completely inhibited. However, many pathologies are linked to an invasive and uncontrolled angiogenesis. Arthritis, for example, is a pathology caused by damage to cartilage caused by invasive neovessels. In diabetic retinopathy, invasion of the retina by neovessels leads to the patients' blindness; neovascularization of the ocular apparatus represents the major cause of blindness and this neovascularization dominates around twenty eye diseases. Lastly, the growth and metastasis of tumors are linked directly to neovascularization and are dependent on angiogenesis, and the tumor itself stimulates the growth of the neovessels. Moreover, these neovessels present escape pathways, allowing metastatic tumor cells to reach the blood circulation and cause metastases in remote sites such as the liver, lungs and bones.
In other pathologies such as cardiovascular diseases, peripheral arterial diseases, and vascular and cerebral lesions, angiogenesis can present an important therapeutic base. The promotion of angiogenesis in the damaged sites can lead to the formation of blood neovessels lateral and alternative to the damaged vessels, thereby providing blood flow and, by consequence, oxygen and other nutritive factors required for the survival of the tissues in question.
The formation of neovessels by endothelial cells involves the migration, growth and differentiation of endothelial cells. The regulation of these biological phenomena are directly linked to gene expression. In the area of angiogenesis, a constantly growing number of studies show that the regulation of angiogenesis is implemented via an equilibrium between the factors acting directly on the endothelial cell. These factors can be angiogenic stimulants, on the one hand, such as, among others, VEGF, FGFs, IL-8, HGF/SF and PDGF. They can also be angiogenic inhibitors such as, among others, IL-10, IL-12, gro-α and -β, platelet factor 4, angiostatin, the inhibitor derived from human chondrocyte, thrombospondin and the leukemia inhibitory factor. (Jensen, Surg. Neural., 1998, 49, 189-195; Tamatani et al., Carcinogenesis, 1999, 20, 957-962; Tanaka et al., Cancer Res., 1998, 58, 3362-3369; Ghe et al., Cancer Res., 1997, 57, 3733-3740; Kawahara et al., Hepatology, 1998, 28, 1512-1517; Chandhuni et al., Cancer Res., 1997, 57, 1814-1819; Jendraschak and Sage, Semin. Cancer Biol., 1996, 7, 139-146; Majewski et al., J. Invest. Dermatol., 1996, 106, 1114-1119).
One of the mechanisms by which cells respond to external stimulus is the recruitment of chains constituted by a set of proteins which provide for the relay of an external signal to the interior of the cells. By providing for the transduction of the extracellular signal, this chain changes the intracellular environment thereby controlling gene transcription (reviews: Avruch, 1998, Mol. Cell. Biochem., 182, 31-48; Karin, 1998, Ann. NY Acad. Sci., 851, 139-146). A large number of these protein chains, and by consequence the signal pathways which are highly conserved via evolution, are collectively designated the pathways of the “mitogenic agent activated protein kinases” (MAPK) (Gupta et al., 1996, EMBO J., 15, 2760-2770; Madhani and Fink, 1998, Trends Genet. 14, 151-155). The classic MAPK pathway is triggered by the binding of the growth factors to their receptor on the cell surface leading to the activation of the protein Ras, which is a GTPase. This pathway results in the activation of the protein kinases regulated by extracellular signals (ERKs), leading to gene transcription and cell proliferation. A parallel MAPK pathway is stimulated by stress factors such as osmotic shock, cytotoxic products, UV radiation or inflammatory cytokines. This pathway results in the activation of the stress-activated protein kinases known by the designation of kinases acting on the N-terminal of c-Jun (SAPK/JNKs) (Karin, 1998, Ann. NY Acad. Sci. 851, 139-146). A second stress-activated pathway leads to the activation of MAPK p38. The effect of stress activation extends to the proliferation, differentiation and even the gene transcription leading to the termination of this cellular cycle and/or apoptosis, depending on the cell type and the stimulus (Karin, 1998, Ann. NY Acad. Sci. 851, 139-146).
Many studies have reported a role for MAPK 1 and 2 as well as MAPK p38 in the transduction pathway of the signal induced by the angiogenic or anti-angiogenic factors during angiogenesis, but no role has been reported for MAPK4 in this process (Tanaka et al., 1999, Jpn. J. Cancer Res., 90: 647-654; Erdreich-Epstein et al., 2000, Cancer Res., 60: 712-721; Gupta et al., 1999, Exp. Cell Res., 247: 495-504; Bais et al., 1998, Nature, 391: 24-25; Rousseau et al., Oncogene, 1997, 15: 2169-2177; Shore et al., 1997, Placenta, 18: 657-665).
MAPK 4 is one of the members of the MAPK family. This kinase phosphorylates directly and thereby activates the kinases acting directly on the N-terminal c-Jun (JNK) in response to stress and/or inflammatory cytokines. MAPK 4 is expressed in different tissues, however there is seen an abundance of expression of this kinase in the skeletal muscles and the brain. Mice deficient in the gene of MAPK 4 develop abnormal hepatogenesis and die in the embryogenic state on the fourth day. However, cell lines deficient in MAP 4 have been obtained. These lines are characterized by the absence of gene transcription dependent of JNK and the transcription factor AP-1. Moreover, T lymphocytes deficient in MAPK 4 exhibit a decoupling of the production of IL-2 subsequent to the activation of the T cell receptors, suggesting a key role for MAPK4/JNK in the inflammatory process. The mutation of MAPK4 in certain carcinomas indicates that it can play a tumor suppressor role. Although the control of the expression and activity of MAPK is currently the object of intense analyses and studies, these studies involve an approach for developing an anti-inflammatory and anticancer therapy. However, the role of MAPK4 in angiogenesis has not been demonstrated.
Pedram et al. (Endocrinology, 2001, 142: 1578-86) showed that the natriuretic peptide suppresses or inhibits the angiogenesis induced by VEGF; they also showed that the activation of the kinases acting directly on the N-terminal of c-Jun is an important state in the induction of angiogenesis by VEGF. In opposition, Jimenez et al., Oncogene, 2001, 20: 3443-3448) reported that the activation of the kinases acting on the N-terminal of c-Jun is necessary for the inhibition of neovascularization by thrombospondin 1.
Neither of these studies reported a specific role of MAP4K4 in the regulation of angiogenesis.
The G proteins (proteins binding guanine) play a major role in the transmembrane signaling pathways by transmission of extracellular signals via the transmembrane receptors to their appropriate intracellular effectors (Gilman, 1987, Ann. Rev. Biochem., 56, 615-649; Simon et al., 1991, Science, 252, 802-808). After binding of the ligand, the receptor catalyzes the exchange of the GDP for a GTP in the alpha subunit of the heterotrimer G protein which induces its activation and the dissociation of the alpha-GTP subunit from the beta and gamma subunits (Gilman, 1987, Ann. Rev. Biochem., 56, 615-649). The G-protein-dependent signaling pathways are designated for amplifying and integrating a multiplicity of both stimulatory and inhibitory responses, and their importance in cell function is such that they are tightly regulated. PhLP (phosducin-like protein) is one of these regulatory elements; it belongs to the family of phosducins and its isoforms, proteins that bind the G protein beta/gamma subunits, thereby blocking their function (Lee et al., 1987, Biochemistry 26, 3983-3990; Miles et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 10831-10835; Craft et al., 1998, Biochemistry 37, 15758-15772). It has been proposed that phosducin, strongly expressed in the photoreceptor cells of the retina (Lee et al., 1987, Biochemistry 26, 3983-3990; Wilkins et al., 1996, J. Biol. Chem., 271, 19232-19237) intervenes in the adaptation to light (Willardson et al., 1996, Proc. Natl. Acad. Sci. USA, 93, 1475-1479). In contrast, the function of PhLP is not as well understood; this protein is even more widely expressed (Miles et al., 1993, Proc. Natl. Acad. Sci. USA 90, 10831-10835) and also binds the G protein beta/gamma subunits with high affinity (Schröder and Lohse, 1996, Proc. Natl. Acad. Sci. USA, 93, 2100-2104; Thibault et al., 1997, J. Biol. Chem., 272, 12253-12256). It has been proposed that this protein represent a phosducin homologue that regulates a certain number of G-protein-dependent pathways in many types of cells (Savage et al., 2000, J. Biol. Chem., Vol. 275, 39, 30399-30407).
However, no role of PhLP has been described to date in the regulation of angiogenesis.
SRp75 belongs to the family of SR proteins due to the fact that it contains in the N-terminal position a conserved domain RRM (RNA recognition motif), a glycine-rich region, an internal region homologous with the RRM and a long (315 aa) C-terminal domain composed essentially of alternating serine and arginine residues (RS domain) (Zahler et al., Mol. Cell Biol. 1993 July; 13(7): 4023-8). The SR proteins constitute a family of nuclear phosphoproteins which are necessary for constitutive splicing but also influence the regulation of alternative splicing. The SR proteins have a modular structure (one or two RRM domains and one RS domain). Each domain in the SR proteins is a functional module. The coordinated action of the RRM domains determines their binding specificity to RNAs, whereas the RS domains function as splice activators (Cáceres et al. 1997, J. Cell Biol., 139, 225-238; Chandler et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 3596-3601; Mayeda et al., 1999, Mol. Cell. Biol., 19, 1853-1863; Graveley and Maniatis, 1998, Mol. Cell, 1, 765-771). Various studies have suggested the unique functions in alternative splicing of the pre-mRNA for the particular SR proteins, especially since they are expressed differentially in a variety of tissues. These SR proteins are thus presented as crucial in the regulation of splicing during cell development and differentiation (Zahler et al., Science 1993 Apr. 9; 260 (5105): 219-222; Fu, 1993, Nature, 365 (6641): 82-8; Cáceres and Krainer, 1997 (ed. Krainer), Oxford University Press, Oxford, UK, pp. 174-212; Valcarcel and Green, 1996, Trends Biochem. Sci., 21(8): 296-301). A recent study showed a variable level of expression of SRp75 in different lymphoid cell lines (Dam et al., 1999, Biochim. Biophys. Acta; 1446(3): 317-33).
To date, no role in the regulation of angiogenesis has been described for either SFRS4 or SRp75 nor for the homologous protein of this factor.
Carboxypeptidase D (CPD of the S10 family of serine peptidases) is a transmembrane protein (180 kDa) which matures the proteins in the trans-Golgi network and notably the proteins secreted via the constitutive pathway such as the growth factors and their receptors: insulin receptor, insulin-like receptor of growth hormones (Reznik et al., 1998; J. Histochem. Biochem., 46, 1359). It is a carboxypeptidase with an activity identical to that of carboxypeptidase E (CPE-like) which is widely distributed in the tissues. The carboxypeptidases intervene in the elimination of basic amino acids from the C-terminal part of the peptide in order to generate either the bioactive product or the precursor for the formation of the C-terminal amide group (Fricker, 1988, Ann. Rev. Physiol., 50, 309-321; Fricker, 1991, (ed.) Peptide Biosynthesis and Processing, pp. 199-230, CRC Press, Boca Raton, Fla.).
CPD is constituted in humans by three carboxypeptidase-like domains, of one transmembrane domain and a small cytosol tail of 58 residues (Novikova et al., 1999, J. Biol. Chem., 274, 28887) capable of binding the phosphatase A protein (PP2A) (Varlamov et al., 2001, J. Cell Science, 114, 311). This is a highly conserved protein among the species with similar enzymatic properties.
CPD is expressed to a high degree in the human placenta. It is found notably in the endothelial cells, the trophoblasts, the amniotic epithelial cells, the chorionic endothelial villus cells and the smooth muscle vascular cells of umbilical cords (Reznik et al., 1998; J. Histochem. Cytochem., 46, 1359). CPE and CPD are also implicated in the production of the precursor of endothelin 1 (ET-1). This suggests that CPE and CPD are implicated in the production of certain umbilical and placental peptides having autocrine and/or paracrine functions.
To data there have been no descriptions of any regulatory role of the protein CPD in angiogenesis.
The protein USP9X belongs to the family of UBPs (ubiquitin proteases), a group of enzymes whose function is to invert the ubiquitination reaction by removing the ubiquitin residue from numerous substrates implicated in cell division, growth, differentiation, signaling or activation of transcription (Liu et al., 1999, Mol. Cell Biol., 4, 3029-38; Zhu et al., 1996, Proc. Natl. Acad. Sci. USA 93: 3275-3279; Verma et al., 1995, Genes Dev. 9: 2723-2735). The ubiquitination of the proteins is an important phenomenon in the regulation of the biological pathways such as transduction activated by cytokines (Baek et al., 2001, Blood, 98, 636-642). The UBPs are characterized by a conserved core domain with surrounding divergent sequences and more particularly to the N-terminal part enabling the specificity of the substrate. These N-terminal divergences can alter the localization and confer multiple functions on the different members of the large family of UBPs (Lin et al., 2001, J. Biol. Chem., 276(23): 20357-63). Certain specific proteases of ubiquitin have already been described or suggested as implicated in certain biological processes such as UBP109 in embryonic development (Park et al., 2000, Biochem. J., 349. 443-53), UBP43 in the differentiation of hematopoietic cells (Liu et al., 1999, Mol. Cell. Biol., 4, 3029-38). The ubiquitination pathway is implicated in angiogenesis (Ravi et al., 2000, 14, 34-44; Sutter et al., Proc. Natl. Acad. Sci. USA, 97, Issue 9, 4748-4753) but USP9X has not been described to date as a regulator of angiogenesis.
The sequence of this mRNA has a coding sequence from nucleotide 136 to nucleotide 3795. There was thus identified a protein GS-P22 resulting from the translation of this mRNA. This protein nardilysine is composed of 1219 aa. It is identified as number SEQ ID No. 74 in the attached sequence listing.
N-arginine dibasic convertase (NRD convertase; nardilysine; EC 3.4.24.61) is a metalloendopeptidase of the family of the insulinases that cleave specifically the peptides (particularly the neuroendocrine peptides such as somatosatin-28, dynorphine-A, the natriuretic atrial factor) on the N-terminal side of an arginine residue at the level of the dibasic sites in vitro (Cohen et al., 1995, Methods Enzymol., 248, 703-716; hospital et al., 1997, Biochem. J., 327, 773-779). Its exact function in vivo still remains poorly understood but many enzymes of the same family are implicated in the maturation of the prohormones and proproteins (Winter et al., 2000, Biochem. J., 351, 755-764). The NRD convertase activity is present principally in the endocrine tissues and to a majority degree in the testicles (Chesneau et al., 1994, J. Biol. Chem., 269, 2056-2061). It can be localized both in the cytoplasm and at the cell surface (Hospital et al., 2000, Biochem. J., 349, 587-597).
At present, there is limited knowledge regarding the regulation of the expression and activity of NRD convertase. The activity appears to be regulated by the amines that bind the acid domain (stretch) of the enzyme (Csuhai et al., 1998, Biochemistry, 37(11): 3787-94). It has also been shown that retinoic acid can modulate the expression of the enzyme in human neuroblast lines (Draoui et al., 1997, J. Neurooncol., 31, 99-106).
In the adult rat, the regulated expression of NRD convertase during spermatogenesis and its concentration in the flagellum suggests a role of this enzyme in the differentiation of male germinal cells (Chesneau et al., 1996, J. Cell Sci. 109, 2737-2745). It has also been proposed that this enzyme plays a specific role in neuronal development (Fumagelli et al., 1998, Genomics 47, 238-245). NRD convertase has recently been described as a new specific receptor of the heparin-binding EGF growth factor (HB-EGF) which controls cell migration (Nishi et al., 2001, EMBO J., 20(13): 3342-50).
In contrast, no role has been discovered to date for NRD1 in the regulation of angiogenesis.
The gene of acute lymphoblastic leukemia-1 (ALL)-1 or myeloid-lymphoid or mixed-lineage leukemia (MLL) or also designated human tri-thorax (HRX) on the human chromosome 11, band q23, is the site of many locally regrouped chromosome alterations (deletions, partial duplications, translocations) associated with various types of leukemia. The structurally variant proteins derived from the altered gene are presumed to cause the malignant transformation of the precursor hematopoietic cells (Nilson, Br. J. Haematol., 1996, 93(4): 966-72; Kobayashi et al., 1995, Leuk. Lymphoma, 17(5-6): 391-9).
The protein MLL is a large nuclear protein with zinc finger motifs and SET domain, highly conserved, of 200 aa localized in the C-terminal part. This protein is expressed to a high degree during embryogenesis; studies have shown that this protein is a positive regulator of the homeobox genes Hox (Yu et al., 1995, Nature (London) 378, 505-508). The protein MLL is described as being implicated in transcriptional maintenance in the development which functions in multiple morphogenetic processes (Yu et al., 1998, Vol. 95, Issue 18, 10632-10636). It has been suggested that the protein MLL plays a role in the regulation both of cell proliferation and survival in the developing embryo (Hanson et al., 1999, Proc. Natl. Acad. Sci. USA, 96, Issue 25, 14372-14377).
To date, there have been no descriptions of any role of the protein MLL in the regulation of angiogenesis.
Since its identification in 1995, the gene ATRX (synonyms XNP, XH2) has been shown to be the gene of numerous forms of diseases; different mental retardation syndromes associated with chromosome X are linked to the mutations of this gene. (Review: Gibbons and Higgs, 2000, Am. J. Med. Genet., 97(3): 204-212.) This gene codes for a protein of the subgroup SNF2 of the superfamily of the helicases and ATPases (Picketts et al., 1996, Hum. Mol. Genet. 5 (12): 1899-907); these domains suggest that the protein ATRX has a role in transcriptional regulation via an effect on the structure and/or the function of chromatin, but its exact role still remains unknown. No role in the regulation of angiogenesis has been described to date.
The transporter of CMP-sialic acid is implicated in the process of maturation of glycosylation and more particularly of sialyation; it enables the translocation of cytosolic CMP-sialic acid through the membrane of the Golgi apparatus required for the sialyation of the membrane or secreted proteins as well as the lipids in this compartment. (Hirschberg and Snider, 1987, Ann. Rev. Biochem., 56, 63-88; Hirschberg, 1996, in Organellar Ion channels and Transporters, Society of General Physiologists, 49th Annual Symposium (Clapham, D. E. and Ehrlich, B. E., eds.), pp. 105-120, Rockefeller University Press, New York). The regulation of the transport of CMP-sialic acid is still poorly understood although an augmentation of sialyation was observed at the surface of tumor cells (Santer et al., 1989, Eur. J. Biochem., 181, 249-260; Saitoh et al., 1992, J. Biol. Chem., 267, 5700-5711; Bresalier et al., 1996, Gastroenterology, 110, 1354-1367; Gorelik et al., 1997, Cancer Res., 57, 332-336) and that the inhibition of the CMP-sialic acid transporter reduces the growth and metastases of tumor cells (Harvey, B. E. and Thomas, P. (1993) Biochem. Biophys. Res. Commun. 190, 571-575). However, to date, no implication of this transporter in the regulation of angiogenesis has been reported.
Cbl-b belongs to the Cbl family, highly conserved among the species. The Cbl proteins are characterized in their N-terminal part by a putative domain binding phosphotyrosines and a RING FINGER motif in the C-terminal part, the Cbl proteins of mammals containing a proline-rich region, conserved tyrosine residues and a zipper leucine motif. The Cbl proteins participate in the signaling of the proteins of the tyrosine kinase receptors as well as the antigens and receptors of cytokines by associating them at their cytoplasm tail providing the continuity of the signal of these receptors. The protein Cbl is recruited by the tyrosine kinase module of these receptors and the phosphorylated tyrosines after cell activation. Cbl functions as a docking protein and associates itself with molecules containing the domains SH2 and SH3, including the family of the adapters Crk and Vav. It has been proposed that the Cbl proteins are negative regulators of the signaling of the tyrosine kinase receptors (Smit et al., Crit. Rev. Oncog. 1997; 8:359-79) as well as positive modulators of the signalization of receptors such as the superfamily of the TNF receptors (Arron et al., J. Biol. Chem. 2001 Aug. 10; 276: 30011-7).
The protein Cbl-b, expressed at high levels in many tissues and cells including the hematopoietic cells (Keane et al., Oncogene 1995 Jun. 15; 10(12): 2367-77) is implicated in the installation of the lymphocyte activation threshold (Bachmaier et al., Nature 2000, 403: 211-6; Fang et al., J. Biol. Chem. 2001; 276: 4872-8). The regulatory subunit P85 of phosphatidylinositol 3-kinase (P13K) was identified as being its substrate. Cbl-b, by its ligase activity of the protein ubiquitin E3, negatively regulates this regulatory subunit P85 (Fang D, Nat. Immunol. 2001 September; 2(9): 870-5). Cbl-b is also a negative regulator of the signalization of the receptor of the epidermal growth factor, EGFR (Ettenberg et al., Oncogene 1999; 18: 1855-66; Ettenberg et al., J. Biol. Chem. 20011 276: 27677-84).
In contrast, there have been no descriptions to date that Cbl-b plays a role in the regulation of angiogenesis.
The base chromatin unit in eukaryotes is the nucleosome. A nucleosome is constituted by a 146-bp DNA sequence wound around an octamer of proteins, the histones H2A, H2B, H3, H4 (Luger et al., 1997, Nature, 389, 251-260). The heterogeneity in the structure of nucleosomes can be a transcriptional regulation mechanism. This heterogeneity is created either by post-translational modifications of the histones such as acetylation, phosphorylation, methylation, ubiquitination (Mizzen et al., Cold Spring Harb. Symp. Quant. Biol. 1998; 63: 469-81; Workman and Kinston, 1998, Ann. Rev. Biochem., 67: 545-579) or by the incorporation of histone variants in the nucleosome. The different histone variants enable the specialization of the structure of the nucleosome for specific purposes; the specific histone variants of sperm, for example, facilitate the dramatic compaction of DNA which occurs during spermatogenesis (Wolffe, 1998, Chromatin: Structure and Function, 3rd Ed., Academic Press, San Diego; Doenecke et al., 1997, Adv. Exp. Med. Biol., 424, 37-48).
The histone H2A.F/Z is a family of variants of the histone H2A which is highly conserved across the species and substantially divergent from the histone H2A of phase S in given species (Jackson et al., 1996, Trends Biochem. Sci., 221, 466-467; Jiang et al., 1998, Biochem. Biophys. Res. Commun., 245, 613-617). The exact function of H2A.F/Z is still unknown but this histone could play a role in transcriptional regulation because in Tetrahymena its expression is associated with the transcriptionally active micronucleus and in Drosophila its incorporation in the chromatin during development coincides with the beginning of the expression of the zygote gene (Clarkson et al., 1999, DNA Cell Biol., 18, 457-462; Stargell et al., 1993, Genes Dev., 7, 2641-2651).
In contrast, no role is known for the histone H2A.F/Z to date in the regulation of angiogenesis.
Casein kinase II (CKII) is a ubiquitous serine/threonine kinase which is localized both in the cytosol as well as in the nucleus of eukaryote cells. CKII phosphorylates more than one hundred substrates, many of which are implicated in the control of cell division and in the transduction of the signal (review: Allende and Allende, 1995, The FASEB Journal, Vol. 9, 313-323). CKII exists in a tetramer form composed of two alpha and/or alpha′ catalytic subunits and two regulatory subunits (beta). The beta subunits appear to act so as to stabilize the alpha and/or alpha′ subunits and also influence the specificity of the substrate and the kinetic properties of the enzyme (Dobrowolska et al., 1999, Mol. Cell. Biochem., 191(1-2): 3-12). Certain studies have shown that the activity of casein kinase is stimulated by growth hormones or factors such as insulin, IGF-I, EGF (Sommercorn et al., 1987, Proc. Natl. Acad. Sci. USA, 84, 8834-8838; Klarlund et al., 1988, J. Biol. Chem., 263, 1872-1875; Ackerman and Osheroff, 1989, J. Biol. Chem., 264, 11958-11965), this activation resulting from an augmentation of the phosphorylation of the beta subunit of casein kinase (Ackerman et al., 1990, Proc. Natl. Acad. Sci. USA, 87, 821-835). Various studies have shown a deregulated expression of CKII in tumors. Recent studies have demonstrated that the overexpression of CKII in tumor cells is not solely a reflection of the proliferation of the tumor cells but also it can reflect the pathobiological characteristics of the tumor. The deregulation of CKII could influence the apoptotic activity of these cells (review: Tawfic et al., 2001, Histol. Histopathol., 16(2): 573-82). This enzyme is described as having a possible role in oncogenesis (Yu et al., J. Cell. Biochem., 1999, 74(1): 127-34).
In contrast, no differential expression of CKII or of its beta subunit during angiogenesis has been described nor has a role in the regulation of angiogenesis been demonstrated to date.
Described recently, hemicentine is a protein of the extracellular matrix of the immunoglobulin superfamily which is implicated in cell attachment and migration on the basal membrane (Vogel et Hedgecock, 2001, Development, 128(6): 883-94). Its role in the regulation of angiogenesis has not been described to date. This protein contains the sequence of fibulin-6 which belongs to the fibulin family, proteins of the extracellular matrix and of the blood, the two members of which that have been the most extensively studied are fibulin 1 and fibulin 2 (Alexande and Detwiler, 1984, Biochem. J., 217, 67-71; Argraves et al., 1990, J. Cell Biol., 111, 3155-3164; Kluge et al., 1990, Eur. J. Biochem., 193, 651-659; Pan et al., 1993, J. Cell Biol., 123, 1269-1277). They interact with the proteins implicated in cell adhesion such as fibronectin, laminin and fibrinogen (Brown et al., 1994, J. Cell Sci., 107 (Pt. 1), 329-38; Tran et al., 1995, J. Biol. Chem., 270(33): 19458-64; Godyna et al., 1995, Matrix Biol., 14(6): 467-77) or endostatin which is an inhibitor of angiogenesis (Sasaki et al., 1998, EMBO J., 17(15): 4249-56) which confers on them a regulatory function in various biological processes. Fibulin 1, for example, has been described as possibly playing a role in the regulation of the neurotrophic activity of the protein precursor amyloid beta (Ohsawa et al., 2001, J. Neurochem.; 76(5): 1411-20), in homeostasis and thrombosis (Tran et al., 1995, J. Biol. Chem., 270(33)L 19458-64).
In contrast, the function of fibulin 6 remains poorly understood and, in particular, no role of this protein has been described to date in the regulation of angiogenesis.
The protein Syne-2 is poorly understood; it was recently described with a homologous protein, the protein Syne-1 (synaptic nuclear envelope-1). The protein Syne-1 is associated with the nuclear envelopes in the cells of smooth, cardiac and skeletal muscle. Syn-1 is described as the first protein found to be associated selectively with the synaptic nucleus and it has been suggested that it is implicated in the formation or maintenance of nuclear aggregates in the muscle junction (Appel et al., 2000, J. Biol. Chem., Vol. 275, Issue 41, 31986-31995). Syn-2 differs from Syn-1 in its distribution and in its level of expression which is weaker. The two homologous proteins exhibit repeated spectrin domains which are present in different proteins implicated in the structure of the cytoskeleton (Yan et al., Science 1993 Dec. 24; 262(5142): 2027-30).
No role of Syn-2 in the regulation of angiogenesis has been described to date.
The gene seladin-1 was recently identified in the human brain. Seladine-1 appears to be an important factor for the protection of cells against the toxicity of the beta-amyloid peptide and oxidative stress (Greeve et al., 2000, J. Neuroscience, 20(19): 7345-7352). These authors suggest that seladine-1 could be implicated in the regulation of survival and cell death and that the diminished expression of this protein in specific neurons could be the cause for the selective vulnerability in Alzheimer's disease.
In contrast, no role in the regulation of angiogenesis has been described to date for seladine-1.
The protein CHD2 belongs to the family of CHD proteins characterized by a chromodomain. The “chromo” (CHRomatin Organization MOdifier) domain is a conserved region of circa 60 amino acids found in a variety of proteins including the HP1 proteins of Drosophila melanogaster, which is linked to heterochromatin; 4 genes of these family have been identified in the human genome: CHD1, CHD2, CHD3 and CHD4 (Woodage et al., 1997, 94, 11472-11477). Chromodomain confers on the protein a role in the compaction of chromatin (Paro, 1990, Trends Genet. 6: 416-421; Singh et al., 1991, Nucleic Acids Res. 19: 789-794; Aasland and Stewart, 199, Nucleic Acids Res. 23: 3168-3173; Koonin et al., 1995, Nucleic Acids Res. 23: 4229-4233; Messner et al., 1992, Genes Dev., 6, 1241-1254; James and Elgin, 1986, Mol. Cell. Biol. 6, 3862-3872). The CHDs contain a second conserved domain, the domain Myb which is implicated in the binding with DNA (Klempnauer and Sippel, 1987, EMBO J., 6: 2719-2725). In addition to these domains, CHD2 contains the domain SNF2, found in the proteins implicated in a variety of processes such as the regulation of transcription (e.g.: SNF2, STH1, brahma, MOT1), repair of DNA (e.g.: ERCC6, RAD16, RAD5), recombination of DNA (e.g., RAD54) (review: Eisen et al., 1995, Nucleic Acids Res., 23(14): 2715-23) and lastly a conserved domain of the helicase superfamily, the “DEAH box helicases”. The helicases are implicated in the unwinding of nucleic acids (Matson and Kaiser-Roger, 1990, Ann. Rev. Biochem. 59, 289-329). It was proposed that the “DEAH box” helicases were implicated in the splicing of mRNAs and in the progression of the cell cycle (Ludgren et al., 1996, Mol. Biol. Cell 7, 1083-1094; Imamura et al., 1998, Nucleic Acids Res., 26(9): 2063-8).
The CHDs could play an important role in the regulation of the transcription of genes (Delmas et al., 1993, Proc. Natl. Acad. Sci., 90, 2414-2418; Woodage et al., 1997, Proc. Natl. Acad. Sci. USA, 94, 11472-11477; Tran et al., 2000, The EMBO Journal, 19, 2323-2331).
A recent study showed that CHD4 is induced when endothelial cells are stimulated by TNF-alpha (Murakami et al., 2000, J. Atheroscler. Thromb.; 7(1): 39-44).
No studies have demonstrated an implication of CHD2 in the regulation of angiogenesis.
The role of the protein BRD2 is unknown. This protein is characterized by two bromo domains. The bromo domain is a conserved region, first identified as a signature of 61-63 amino acids; its function being unknown (Haynes et al., Nucl. Acids Res., 1992, 20, 2603). This domain was subsequently identified in transcription factors, co-activators and other proteins are implicated in the transcription or remodeling of chromatin and its boundaries were extended to 110 amino acids. The increasing number of proteins containing this domain is more than forty (Jeanmougin et al., 1997, Trends Biochem. Sci. 22, 151-153; Winston and Allis, 1999, Nature Struct. Biol. 6, 601-604). Certain proteins have a single copy of the domain while others present two copies of the motif. The protein BRD2 is characterized by two bromo domains which is also the case of the transcription factors BDF1 (Tamkun, 1995, Curr. Opin. Genet. Dev., 5: 473-477) or the protein TAFII250, the largest subunit of the multiprotein complex TFIID implicated in the initiation of transcription (Jacobson et al., 2000, Science, 288(5470): 1422-5). The exact function of this domain remains unknown but it is thought to be implicated in protein-protein interactions and could be important for the assembly or the activity of multiple complex components implicated in the modification of chromatin and the transcriptional control of a large variety of eukaryote genes included those which control growth. A large variety of functions directed on chromatin, including but not limited to phosphorylation, acetylation, methylation, coactivation or transcriptional recruitment characterize the complexes which contain the bromo domains. Their versatility and ubiquity provides diverse, rapid and flexible transcriptional responses (review: Denis, 2001, Frontiers in Bioscience 6, d849-852).
The differential expression of proteins containing a bromo domain has already been demonstrated, notably that of a protein homologous to RING3 kinase whose expression is induced by VEGF or bFGF in endothelial cells. It has been suggested that this protein is a new target of the signalization pathway activated by VEGF and bFGF which enables endothelial cells to enter into the proliferative phase of the angiogenesis process (BelAiba et al., 2001, Eur. J. Biochem., 268(16): 4398-407).
In contrast, there have been no reports of BRD2 being implicated in the regulation of angiogenesis.
Syntaxin 3A belongs to the family of syntaxins/epimorphines which are characterized by a size between 30 and 40 kDa, a highly hydrophobic C-terminal end which is probably implicated in the anchoring of the protein in the membrane and a well conserved central region which appears to be in a coiled-coil conformation. The specific profile of this family is based on the most highly conserved region of the coiled-coli domain. The syntaxins are implicated in the intracellular transport of vesicles and their storage in the plasma membrane. Recent studies suggest that different syntaxin isoforms could interact with a defined group of membrane transport proteins and thereby regulate their transport activity (review: Saxena et al., 2000, Curr. Opin. Nephrol. Hypertens., 9(5): 523-7).
Syntaxin 3A is one of the two isoforms (3A and 3B) identified in humans, stemming from an alternative splicing of the same gene. Augmentation of the expression of syntaxin 3A has already been demonstrated over the course of various biological processes such as the neutrophil differentiation of HL-60 cells or in dentate granule cells during the propagation of synaptic plasticity in the nervous system (Rodger et al., 1998, J. Neurochem., 71(2): 666-675; Martin-Martin et al., 1999, J. Leukoc. Biol., 65(3): 397-406).
In contrast, no increased expression of syntaxin has been reported to date in the course of angiogenesis and thus no implication in the regulation of angiogenesis.
Sharp (SMART/HDAC1 Associated Repressor Protein) is a recently isolated gene (Nagase et al., 1999, DNA Res., 6(1): 63-70). SHARP is a potential repressor of transcription whose repression domain (RD) interacts directly with SMRT and at least 5 members of the NuRD complex (nucleosome remodeling and histone deacetylase activities) including the deacetylase histones HDAC1 and HDAC2. SHARP moreover binds to the ARS coactivator of the RNA of the steroid receptor by an intrinsic domain binding RNA and suppresses the transcription activity of the steroid receptor. In this manner, SHARP has the capacity to modify both the bound and unbound nuclear receptors. The expression of SHARP is itself inducible by steroids, suggesting a simple feedback mechanism for the attenuation of the hormonal response (Shi et al., Genes Dev. 2001 May 1; 15(9): 1140-51. The deacetylase histones (HDAC) were shown to be implicated in the induction of angiogenesis by suppressing the expression of the tumor-suppressor genes (Kim et al., 2001, Nat. Med., 7(4): 437-43).
Nevertheless, no role of the SHARP protein has been described in the regulation of angiogenesis.
The exact role of the proliferation potential-related protein identified is still unknown. It presents as its homologous proteins zinc finger domains known to be implicated in protein-protein interactions. This protein is homologous with a member of the family of the retinoblastoma-binding proteins (pRb), retinoblastoma-binding protein 6, also referred to as RBQ-1 (Sakai et al., 1995, Genomics, 30(1): 98-101). The protein pRb (suppressor of the retinoblastoma tumor) acts for controlling cell proliferation, inhibits apoptosis and induces cell differentiation and does this by associating with a large number of proteins (review: Morris and Dyson, 2001, Adv. Cancer Res.; 82: 1-54).
The proliferation potential-related protein has never been described to date as implicated in the regulation of angiogenesis.
The protein HIP1 (Huntingtin interacting protein 1) is a protein of 116 kDa that binds the protein.
Huntingtin, product of the mutated gene in Huntington's disease (Kalchman et al., 1997, Nat. Genet., 16, 44-53; The Huntington's Disease Collaborative Research Group, 1993, Cell 72, 971-983). HIP1 is predominantly expressed in the brain but is also detected in other tissues (Wanker et al., 1997, Human Molecular Genetics, Vol. 6, 487-495). The function of HIP1 is not yet fully understood but it shares the biochemical characteristics and conserved domains with Sla2p, a protein essential for the function of the cytoskeleton in Saccharomyces cerevisiae (Kalchman et al., 1997, Nat. Genet. 16, 44-53; Holtzman et al., 1993, J. Cell Biol. 122, 635-644). HIP1 also contains a leucine zipper domain and is homologous in its C-terminal part with taline, cytoskeleton protein implicated in cell-substratum and cell-cell interactions (Rees et al., 1990, Nature; 347(6294): 685-9). It has recently been shown that the protein HIP1 is implicated in apoptosis (Hackam et al., 2000, J. Biol. Chem., Vol. 275, Issue 52, 41299-41308).
No role of HIP1 in angiogenesis has been described to date.
Nucleoporin 88 (Nup88) is a protein of the nuclear membrane probably implicated in nucleocytoplasmic transport (Formerod et al., 1997, EMBO J., 16: 807-816; Fomerod et al., Oncogene 1966, 13: 1801-1808). Nup88 was found to be associated with the central domain of CAN/Nup214, a component of the complex of a nuclear pore probably implicated in the importation of nuclear proteins, the exportation of nuclear mRNAs and the regulation of the cell cycle (van Deursen et al., 1996, EMBO J., 15: 5574-5583). Nup88 has been shown to be widely distributed and overexpressed in cancerous and fetal cells and tissues (Martinez et al., 1999, Cancer Research 59, 5408-5411; Gould et al., 2000, American Journal of Pathology, 157: 1605-1613).
Nup88 to date has never been described as implicated in the regulation of angiogenesis.
The FKBPs (FK506 binding proteins) are major proteins binding the immunosuppressive drug FK506 with high affinity in vertebrates (1990, Tropschug et al., Nature 346: 674-677; Stein, 1991, Curr. Biol. 1: 234-236; Siekierka et al., 1990, J. Biol. Chem. 265: 21011-21015). Many members of the FKBP family have been identified in various tissues and various cellular compartments, the best known being FKBP12, a cytoplasm isoform (Galat and Metcalf, 1995, Prog. Biophys. Mol. Biol. 63, 67-118; Kay, 1996, Biochem. J. 314, 361-385). The FKBPs belong to a large family cis-trans peptidyl propyl isomerases (PPIase or rotamase). PPIase is an enzyme that accelerates the folding of the protein by catalyzing the cis-trans isomerization of the peptide bonds involving the proline residue (Fischer and Schmid, 1990, Biochemistry 29: 2205-2212). The FKBPs are known to be implicated in many cellular processes such as cellular signaling, protein transport and transcription. Studies of interruption of the gene coding for the FKBPs in plants and animals have emphasized the importance of this family of proteins in the regulation of cell division and differentiation (review: Harrar et al., 2001 Trends Plant Sci.; 6: 426-331). However, despite the fact that they bind surface receptors and despite their Ppiase activity, their physiological function has yet to be defined. It was recently proposed that the Ppiases play a role in the functional rearrangement of the components in the heterocomplex receptors (Schiene-Fisher and Yu, 2001, FEBS Lett., 495(1-2): 1-6).
It has not been claimed to date that the FKBP proteins are implicated in the regulation of angiogenesis.
The complementary DNA (cDNA) of the SALF protein (stoned B/TFIIA-alpha/beta-like factor) was recently identified from a bank of cDNA originating from the human placenta (Ashok et al., 1999, J. Biol. Chem., Vol. 274, Issue 25, 18040-18048). The role of SALF has yet to be identified but this cDNA, characterized as rare, is identical to the sequence of the ALF protein with a more extended N-terminal sequence containing a domain homologous to the Stone B protein of Drosophila (Andrewe et al., 1996, Genetics 143, 1699-1711) and to the adaptor proteins of Clathrina, μ1 (AP47) and μ2 (AP50) (Thurieau et al., 1988, DNA (NY) 7, 663-669; Nakayama et al., 1991, Eur. J. Biochem. 202, 569-674). The protein ALF (TFIIA-alpha/beta-like factor) is also a new factor which is a functional homolog of the transcription factor TFIIA alpha/beta which, with the factor TFIIA gamma, can stabilize the interactions of the element TBP (TATA-binding protein)-TATA and maintain the in vitro transcription dependent of RNA polymerase II. The protein ALF is described as a general transcription factor specific of the testicles (Ashok et al., 1999, J. Biol. Chem. Vol. 274, Issue 25, 18040-180048).
The domain of Stoned B/clathrin AP-like homology of SALF also reveals a potential function in the dependent transport of clathrin in the membrane proteins.
Chen et al. (2001, Biochem. Biophys. Res. Commun. 23; 281(2): 475-482) showed that the gene of the factor SALF is induced by retinoic acid (or the retinoids) in cultured smooth muscle cells.
In contrast, no implication of SALF in the regulation of angiogenesis has been reported to date.
The recently described protein P29 interacts with the protein GCIP (Grap2 cyclin-D interacting protein), itself interacting with cyclin D and the protein Grap2 (Chang et al., Biochem. Biophys. Res. Commun. 2000 Dec. 20; 279(2): 732-7). Grap2 is an adaptor protein specific of the leukocytes of the immune tissues (Qiu et al., 1998, Biochem. Biophys. Res. Commun. 253, 443-447). The adaptor proteins play an essential role in the formation of intracellular signalization complexes relaying the extracellular signals from the plasma membrane to the nucleus of the cell. Grap2 is a central linking protein in the signalization and activation of the immune cells. The type D cyclins respond to the extracellular mitogenic signals (Sherr, 1993, Cell 73, 1059-1065). The protein GCIP, which is expressed in a ubiquitous manner in all human tissues, interacts with Grap2 and the D cyclins. Its expression regulates the phosphorylation of the retinoblastoma (Rb) protein and thereby the transcription pathway controlled by the transcription factor E2F1 belonging to a family of factors that play a major role in the proliferation, differentiation, apoptosis and progression of the cell cycle (Nevins, 1998, Cell Growth Differ. 9, 585-593; Chellappan et al., 1998, Curr. Top. Microbiol. Immunol. 227, 57-103; Dyson, 1998, Genes Dev. 12, 2245-2262). It has therefore been suggested that GCIP plays a role in the control of cell proliferation and differentiation via the signalization pathways controlled by the Grap2 and cyclin D proteins (Xia et al., 2000, J. Biol. Chem., 275(27): 20942-8). The protein P29 which has been localized in the nucleus and is present in multiple tissues has been found to be associated with GCIP and therefore could be implicated in the functional regulation of GCIP (Chang et al., Biochem. Biophys. Res. Commun. 2000 Dec. 20; 279(2): 732-7).
The protein P29 thus appears to play a role in the signaling pathways implicated in cell proliferation and differentiation. However no role in the regulation of angiogenesis has been demonstrated to date either for the protein P29 or for the identified homologous protein.
The gene of TMEM2 has recently been described (Scott et al., Gene 2000; 246(1-2): 265-74); based on its structure, it was suggested that the coded protein TMEM2 was transmembranous. The presence of the RGD motif suggesting a role in cellular adhesion is an implication in the cellular signalization pathways.
No role has been described to date for this protein and in particular no role in the regulation of angiogenesis has been demonstrated.
Although few studies to date have focused on the protein called Dorfin, its gene was recently identified. This gene is ubiquitously expressed in many organs. It binds specifically to the conjugating ubiquitin enzymes, UbCH7 and UbCH8, via its RING-FINGER/IBR domain. Dorfin is proposed as a new member of the RING Finger type ubiquitin ligases. This protein is localized in the centrosome and probably acts in the organization centers of the microtubules (Niwa et al., 2001, Biochem. Biophys. Res. Commun., 281(3): 706-13). No differential expression of Dorfin nor a role in the regulation of angiogenesis have been reported to date.
The protein TM4SF2 belongs to the type 4 transmembranous superfamily certain of whose members are known to be implicated in angiogenesis such as CD9 and PETA-3/CD151 (Klein-Soyer et al., 2000, Arterioscler. Thromb. Vasc. Biol., 20: 360-9; Sincock et al., 1999, J. Cell. Science, 112, 833-844). In contrast the function of TM4SF2 remains unknown. This protein is homologous with the protein TALLA-1, proposed as a specific surface marker of neuroblastomas and neuroblastic leukemia (Takagi et al., Int. J. Cancer, 1995, 61(5): 706-15).
No role for the protein TM4SF2 in angiogenesis has been reported to date.
The ecto-ATPases (ATPases of the cell surface) are enzymes that are ubiquitous in the cells that hydrolyze extracellular ATP and ADP into AMP (review: Plesner, 1995, Int. Rev. Cytol., 158: 141-214). The presence of the ATPDases was demonstrated in the aortic endothelial cells and the smooth muscle cells and described as possibly playing a regulatory role in homeostasis and platelet reactivity by hydrolyzing ATP and ADP (Robson et al., 1997, J. Exp. Med., 185(1): 153-163). Vascular ATPDase is closely homologous to the glycoprotein CD39 whose accession number in the GENBANK database is S73813 and which is identified by the number SEQ ID No. 290 in the attached sequence listing, activation antigen of lymphoid cells, also expressed by human endothelial cells (Kaczmarek et al., 1996, J. Biol. Chem., 271, 51, 33116-33122). Recently, Goepfert et al. (2001, Circulation, no. 104 (25): 3109-3115), implanting bodies containing Matrigel in mutant mice characterized by deficiency of expression of CD39, showed the absence of formation of neovessels around the implanted bodies. These observations led the authors to suspect a role for CD39 in the angiogenesis phenomenon. However, the experimentation described supports a poorly defined role of CD39 in angiogenesis rather than a direct and incontestable role. In fact, due to the fact that mutant mice deficient in CD39 exist, it is possible that embryos of CD39-deficient mice could develop. However, without angiogenesis there is no possible embryonic development. Consequently, either CD39 has a role in angiogenesis and therefore there would not exist viable CD39-deficient mutant mice, or CD39 has no role in angiogenesis. In another article, Goepfert et al. (2001, Circulation, 104 (25): 3109-15) did not demonstrate the absence of expression of CD39 in the mutant mouse endothelial cells employed.
In sum, the study by Goepfert et al. (2001, Circulation, 104(25): 31309-15) does not provide proof of any role of CD39 in angiogenesis.
Control of angiogenesis thus represents a strategic axis, both for basic research (in order to improve our comprehension of numerous pathological phenomena linked to angiogenesis) and for the development of new therapies intended for treating pathologies linked to angiogenesis.
In order to control angiogenesis, many pharmaceutical groups have developed therapeutic strategies based directly on the use of paracrine signals as stimulatory and inhibitory factors as agents for promoting or inhibiting angiogenesis. These strategies are based essentially on the use of these factors in their polypeptide form as stimulatory or inhibitory agents of angiogenesis, or more recently in the form of expression vectors coding for the selected factors.